Tol1-based transgenesis vector

pDon122: A vacant donor plasmid.pDon123: Donor plasmid carrying the GFP gene.pHel105: Helper plasmid. Its vector portion is pCS2+, having the CMV promoter for in vivo expression of the transposase gene and the SP6 promoter for in vitro synthesis of the transposase mRNA.pHel106: A defective helper which is useful for negative control experiments especially when you want to know the net transformation efficiency.

Other: Background Donor and helper plasmids for transgenesis in vertebrates. The donor plasmid contains terminal regions of the Tol1 element and multicloning sites for integration of a gene to be transferred to the host chromosome. The helper plasmid carries the transposase gene of the Tol1 element. Tol1 is a DNA transposon identified in the medaka fish and demonstrated to be active in various vertebrate species. Protocol Recovering plasmid DNA1. Cut out one of the circles of the paper and immerse it in water or TE in a microfuge tube. Other circles are for backup.2. Mix by tapping.3. Centrifuge for 1 minute at >10 krpm.4. Transform competent bacterial cells (commonly used strains, such as JM109, DH5a and XL1-Blue) with a small amount of supernatant.5. Spread the bacteria on an LB/agar plate containingl ampicillin, and incubate the plate at 37 oC for >12 hours.6. Pick up a single colony.7. Amplify bacteria in liquid media.8. Extract plsmid DNA by the standard method.